1. Field of the Invention
The present invention relates to a method for culturing the Langerhans islets suitable for transplantation. More particularly, the present invention relates to a culturing method by which the Langerhans islets can be proliferated in volume, and the fact that proliferated islet autotransplantation can stimulate islet regeneration via islet replication and neogenesis, leads to a perfect diabetes cure.
2. Description of the Prior Art
Diabetes mellitus (usually referred to simply as diabetes) is a complex disease characterized by a grossly abnormal pattern of carbohydrate metabolism resulting from impaired insulin secretion and/or effectiveness. The incidence of diabetes in industrialized countries is about 10%. Indeed, diabetes is the most common serious metabolic disease in the world, it affects hundreds of millions.
Diabetes may be classified as insulin-dependent diabetes or noninsulin-dependent diabetes. An absence of or insufficient intrinsic insulin is a characteristic of insulin-dependent diabetes. Some diabetics have a normal or even higher than normal level of insulin in their blood, but they are quite unresponsive to the hormone. This form of the disease, known as non-insulin-dependent diabetes, typically develops later in life than does the insulin-dependent form. However, the diabetes-causing mechanism with which these two types can be discriminated has yet to be revealed.
For treatment, insulin-dependent diabetics should continue to receive exogenous insulin because their capacity of producing insulin is greatly lowered. However, it is virtually impossible to continuously and properly provide insulin in response to patient""s physiological demands. What is more difficult, the body has an insulin concentration gradient such that the insulin concentration is decreased in order of: the hepatic portal vein, the liver, the hepatic vein, the aorta and the muscle, but an injection of exogenous insulin does not result in such a concentration gradient, which then causes side effects.
The xcex2-cells of the Langerhans islets secrete insulin and 11 other materials. Thus, an injection of only insulin can decrease the blood glucose level, but cannot prevent glucopenia and other complications. Since one of the objectives in the treatment of diabetes is to lower the blood glucose level, blood glucose lowering agents are often employed. These lowering agents, however, should not be prescribed for an extended period of time because they result in resistance. Moreover, blood glucose lowering agents were found to cause serious side effects.
Insulin, as mentioned above, is able to lower blood glucose level as well as gives much lower resistance than do blood glucose lowering agents. However, the necessary amount of insulin varies with a patient""s conditions so that it is very difficult to timely administrate proper dosage of insulin. Upon improper administration of insulin, anti-insulin antibodies may be formed, making diabetes worse.
For curing diabetes, tissue transplantation has recently been of great interest. For example, the pancreas or Langerhans islets are transplanted into a patient who suffers from diabetes to provide a controlled amount of insulin which is necessary for the patient.
In such cases, however, immune rejection is always problematic and must be considered. When the immune rejection occurs, immune suppressors are administered to the patients. In addition, the number of donors are not sufficient relative to the demand.
The treatment of diabetes by insulin administration was first conducted in 1921 by Banting and Best, but they failed to cure the disease because of a diabetic complication. In 1966, Lillihei of Minnesota University first transplanted a portion of the pancreas into a diabetic patient. By 1977, 57 patients had been subjected to the transplantation. However, less than 10% of them survived for one year or more. Recent development of immune suppressors has increased the survival rate of pancreas or kidney transplant recipients up to 70%. For Langerhans islet transplantation, the survival rate amounts up to 90%.
The first thing into which account is taken is the histocompatibility between donor and recipient. If tissue transplantation is performed between two persons who have different histocompatibility, an immune rejection occurs, leading to the destruction of the transplanted islets at the worst. Generally, 50 donors are needed to discover the necessary histocompatibility for one recipient. If fresh islets are transplanted, a large quantity of fibrous tissues grow out from freshly isolated Langerhans islets and divide and surround them if transplanted, so that the ability of the xcex2-cells to secrete insulin in response to a stimulus declines greatly.
In approaching the present invention, the present inventors considered the following:
First, in order for cells or cell groups to proliferate in vitro, they must contain stem cells or progenitor cells therein and be in undifferentiated states. Fortunately, since many stem cells or progenitor cells exist in Langerhans islets, it is highly possible to proliferate undifferentiated Langerhans islets in vitro.
Next, MHC class II antigens, which cause immune rejection, must be absent in the proliferated Langerhans islets and thus, if the blood cells, rich in MHC class II antigens, are eliminated from Langerhans islets, the immune rejection can be greatly reduced in the islet allotransplantation. The longer the islets remain in the culture and proliferate, decreases the immune rejection response.
Finally, fibrous tissues are developed from the crude islets and must be able to be easily removed from the in vitro proliferated islet.
Taking advantage of the above three points, the present inventors tried to proliferate in vitro the Langerhans islets with the aim of preparing them so as to be easily and successfully transplanted in the host for the long term treatment of diabetes.
When the Langerhans islets were grown in a monolayer culture method, they proliferated at a rate of, at most, 80%. However, they poorly secreted insulin so that it was impossible to control the level of the blood glucose. The islets should proliferate at least 5-fold for successful transplantation.
Intensive and thorough research repeated by the present inventors resulted in the discovering that upon in vitro culture in media containing various biochemical materials, the Langerhans islets isolated from rats proliferate at high rates sufficient to be applied for transplantation, release the blood cells from themselves so as to greatly reduce the immune rejection, and function well enough so as to successfully continue to secrete insulin after transplantation according to the present invention.
Therefore, it is an object of the present invention to provide a method for proliferating the Langerhans islets in a suitable state for transplantation, whereby a greatly enhanced treatment effect for diabetes can be brought about.
In accordance with the present invention, there is provided a method for proliferating the Langerhans islets, in which a culture medium is supplemented with radical scavengers, growth factors, a matrix material, nerve growth factor, cell migrating/scattering factors (such as HGF) antinecrosis factors or antiapoptosis factors (such as IGF 1, IGF 2, VeGF) and a cytoskeleton activator (anti-integrin xcex21 antibody) at proper culture times and the proliferation is conducted for an extended period of time, so that the Langerhans islets are depleted of the blood cells and also proliferate sufficiently in order to be suitable for transplantation.
The present inventions are directed to a method for in vitro culturing and proliferating isolated Langerhans islets endocrine cells so as to be suitable for transplantation. To initiate the proliferation viable Langerhans islets endocrine cells including cells capable of differentiating into insulin producing cells are collected and placed in a first culturing medium comprising a basal medium supplemented with serum, at least one radical scavenger selected from the group consisting of nicotinamide, mannitol or superoxide dismutase; at least one growth factor selected from the group consisting of: insulin transferrin selenite-complex (ITS-complex), epidermal growth factor (EGF), platelet derived growth factor (PDGF), thrombin, Linoleic Acid-BSA, hydrocortisone and progesterone, and at least one antinecrosis or antiapoptosis factor selected from the group consisting of. IGF 1, IGF 2, VeGF and culturing the Langerhans islets endocrine cells including cells capable of differentiating into insulin producing cells for a period of about one day in the first culturing medium to form a first culture growth. The first culture growth is collected and incubated at room temperature in fresh DMEM or serum free basal medium with anti-integrin xcex21 antibody for 45xcx9c120 minutes to form a second culture growth.
The second culture growth is then suspended in a matrix material to provide a 3-dimensional culture growth environment and a second culturing medium comprising the supplemented basal medium and further including, at least, another growth factor is added thereto and then culturing proceeds for 1 or 2 days to provide a third culture growth dispersed in the matrix material.
A third culturing medium for culturing the third culture growth in the matrix material is provided and comprises the supplemented basal medium but without VeGF, and optionally adding to the third culturing medium NGF and HGF if the islets of the third culture growth appeared thick and the center of the islets appeared dark, or optionally adding to the third culturing medium NGF and anti-integrin xcex21 antibody if the islets appeared too spread out and then culturing for a period of about one or two days to form a fourth culture growth. The islets are then collected from the matrix material, placed in a suitable vessel and an enzyme such as dispase is added to the collected islets and to loosen or enable removal of any adhering gel and then incubated for about 10 minutes to provide an incubated product. The incubated product is then aspirated back and forth numerous times causing the gel acted on by the dispase to be removed from the islets thereby exposing the fibroblasts to the force created during the back and forth aspiration which appears to cause the fibroblasts to become separated from the surface of the islets to prepare fibroblast free islets.
The above process takes about 7 days and can be repeated as follows. A fourth culturing medium comprising the basal medium supplemented with serum, insulintransferrin-sodium selenite (ITS), Linoleic Acid-BSA, thrombin, EGF, nicotinamide, VeGF, IGF-1, IGF-2, superoxide dismutase and mannitol is provided and then the fibroblast free islets are cultured for about 8-12 hours to provide a fifth culture growth. The fifth culture growth is collected and cultured in a fifth culturing medium comprising DMEM with anti-integrin xcex21 antibody for 45xcx9c120 minutes at room temperature to form a sixth culture growth. The sixth culture growth is then suspended in a matrix material to provide a 3-dimensional culture growth environment and a sixth culturing medium comprising the supplemented basal medium and further including, at least, another growth factor is added thereto and then cultured for 1 or 2 days to provide a seventh culture growth dispersed in the matrix material.
A seventh culturing medium which comprises the supplemented basal medium without VeGF, and optionally adding to the third culturing medium NGF and HGF if the islets of the third culture growth appeared thick and the center of the islets appeared dark, or optionally adding to the third culturing medium NGF and anti-integrin xcex21 antibody if the islets appeared too spread out is provided for culturing the seventh culture growth dispersed in the matrix material for a period of about one or two days to form an eighth culture growth. The islets are then collected from the matrix material and an enzyme, such as dispase, is added to the collected islets and to any adhering gel and incubated for about 10 min. to provide an incubated product. The incubated product is aspirated back and forth numerous times causing the gel acted on by the enzyme to be removed form the islets thereby exposing the fibroblasts, if any, to the force created during the back and forth aspiration causing the fibroblasts to become separated from the surface of the islets to prepare an increased number of fibroblast free islets.
In the present method it is preferred that the serum used in the medium is obtained from the same species as that of the Langerhans islets to be proliferated. Thus, where the Langerhans islets are from a rat or human, the serum used is also rat, human serum, respectively, preferably the percent of serum in the medium is about 10%. The growth factor added to the second and sixth culturing medium is preferably pituitary extract.
The method of the present invention also includes using viable Langerhans islets endocrine cells including cells capable of differentiating into insulin producing cells for proliferation which are derived from a patient for proliferation according to the present invention which are then used for autotransplantation back into the same patient. This results in regeneration of the islets in the patient as described below.
The present invention also includes a method for removing fibroblasts growing from the surface of in vitro proliferated Langerhans islets by providing a plurality of proliferated islets having fibroblasts growing therewith in a gel matrix. The islets are collected from the gel matrix, along with the fibroblasts which are growing with the islets. An enzyme, such as dispase, is added to the collected islets and to any gel adhering to the surface of the collected islets and then this is incubated to provide an incubated product. The incubated product is then aspirated back and forth numerous times causing the gel acted on by the dispase to be removed from the islets thereby exposing the fibroblasts to the force being created during the back and forth aspiration which causes the fibroblasts to become separated from the surface of the islets to prepare fibroblast free islets.
The present invention also includes the culture product of proliferated fibroblast free islets produced by the method according to the present invention and the use of the proliferated fibroblast free islets produced by the method according to the present invention in treating diabetes mellitis by transplanting the proliferated Langerhans islets endocrine cells into a patient suffering from diabetes mellitis. In addition, the present invention further includes the use of the proliferated fibroblast free islets from a patient produced by the method according to the present invention to treat diabetes mellitis and to regenerate islets in the patient by autotransplanting the proliferated Langerhans islets endocrine cells into the patient suffering from diabetes mellitis.